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Einrichten des Wasser-Labyrinth <ol><li> Die Hauptkomponente des Wassers Labyrinth einzurichten sollte ein runder Pool, ca. 6 Meter im Durchmesser und ca. 3 Meter tief. Wenn Sie die Aufnahme der Aufgabe sind mit einer Videokamera sicher, dass alle Seiten des Labyrinths sind im Sichtfeld der Kamera zu sehen. </li><li> Füllen Sie das Wasser Labyrinth mit Leitungswasser, das fast 26 sein sollte ° C. Dies kann mehrere Stunden dauern, so sollte frühzeitig erfolgen. Überprüfen Sie regelmäßig die Wassertemperatur, so dass es innerhalb eines Grades von 26 ° C </li><li> Legen Sie die Escape-Plattform in der Mitte des Pools. Während des Trainings muss ausgesetzt werden, einen Zoll über dem Wasser. Dies lehrt die Ratte, dass es eine Plattform, und dass es den Weg raus aus dem Wasser. Später, nachdem das Tier trainiert und zum Testen bereit ist, wird die Flucht-Plattform direkt unter der Oberfläche des Wassers, und wird nicht sichtbar, da das Wasser undurchsichtig mit Milch oder ungiftige Farbe sein. Jetzt wird das Wasser Labyrinth bereit für das Training der Tiere. </li></ol> Pre-Training für die Wasser-Labyrinth <ol><li> Für die Wasser-Labyrinth Ausbildung, die Plattform sollte in der Mitte des Pools und belichtet einen Zoll über der Oberfläche, so dass die Tiere weiß, dass es da ist. Das Wasser sollte innerhalb eines Grades von 26 ° C werden </li><li> Jedes Tier wird durchlaufen drei aufeinander folgenden Versuchen. Zuerst legen Sie das Tier auf der Plattform für 20 Sekunden. </li><li> Das Wasser Labyrinth hat 4 Startpositionen: Norden, Süden, Osten oder Westen. Nehmen Sie das Tier auf eine dieser Positionen. Lower Tier ins Wasser, indem Sie es mit der Hand und brachte es vorsichtig nach unten ins Wasser tail-Ende zuerst. Kein Stress der Tiere durch Eintauchen in Kopf zuerst. </li><li> Lassen Sie das Tier schwimmen / search für die Plattform für maximal 60 Sekunden. Im ersten Moment mag das Tier am Rand des Pools schwimmen auf der Suche nach einem Ausweg. Irgendwann wird das Tier lernen, für die Plattform suchen und klettern. </li><li> Nachdem das Nagetier erreicht die Plattform, stoppen Sie den Timer, und notieren Sie die Zeit. Wenn es nicht finden die Plattform in 60 Sekunden, dann nehmen Sie die Zeit für diese Studie als 1 Minute. Heben Sie nicht das Tier, wenn es um die Plattform zu erreichen schlägt fehl. Teach das Tier, es muss auf die Plattform zu schwimmen. Daher sanft in das Tier auf die Plattform mit der Hand. Lassen Sie das Tier auf der Plattform sitzen für 15 Sekunden. Wenn es oder fällt springt, sanft in sie zurück. Dies wird Zug das Tier, es muss auf die Plattform, um aus dem Pool gerettet werden bleiben. </li><li> Wiederholen Sie diesen Vorgang für zwei weitere Versuche, beginnend bei einer anderen Richtung für jeden Versuch. </li><li> Sobald das Tier alle drei Studien abgeschlossen hat, trocknen Sie es mit einem Handtuch. Wiederholen Sie die drei Probetraining Prozess für alle Tiere nacheinander. Halten Sie die Richtungen die gleichen für alle von ihnen, und ihre Zeiten erfassen. </li><li> Nun, da die Tiere darauf trainiert sind, sind sie bereit, um das Wasser Labyrinth-Test durchzuführen. </li></ol> Wasser Labyrinth Test <ol><li> Zu Beginn experimentelle Studien mit dem Wasser Labyrinth, tanken, so dass die Plattform ist ein Zoll unter der Oberfläche des Wassers. Verwenden Sie fettfreie Trockenmilch, oder 125 Milliliter ungiftigen weißen Tempera zu malen, machen das Wasser undurchsichtig. </li><li> Die Beleuchtung und Wassertemperatur sollte der gleiche wie in den Trainingsprozess werden. Jedes Tier wird durchlaufen 12 Studien, die die 3-Studien werden für jeden ab Richtung. Jeder Versuch dauert 60 Sekunden. Bevor Sie beginnen, wählen Sie die Reihenfolge der Ausgangspunkt Richtungen. Verwenden Sie nicht den gleichen Start Richtung zweimal in Folge, und auch nicht wiederholen der gleichen Reihenfolge für jede der Richtungen </li><li> Mit Blick auf die Wand des Pools, wird das Tier-Handler das Tier im Wasser statt, und um einen Schritt zurück aus dem Pool und sitzen in einem bestimmten Ort, während das Tier führt das Labyrinth Aufgabe. Ein tierisches Verhalten Tracking-System wie das SMART-System (San Diego Instruments) können verwendet werden, um den Weg, wie auch andere Variablen zu überwachen. Videoaufnahmen jeder Studie ist ein preiswerter, und perfekt geeignet, alternative. </li><li> Überwachen des Tieres, bis er die Plattform erreicht, und notieren Sie die Zeit, die. Wenn das Tier nicht erreicht die Plattform in 60 Sekunden wird der Hundeführer es um die Plattform zu führen, wie in der Ausbildung. So oder so, lassen Sie das Tier für 10 Sekunden zu sitzen, und dann trocknen sie ab und gibt das Tier in einen Betrieb Käfig. </li><li> Fahren Sie mit dem jedes Tier für alle 12 Versuche, mit dem Tier-Handler wieder in die gleiche vorgesehenen Stelle bei jedem Versuch. Die Reihenfolge der Tests sollte: Versuch 1 für alle Tiere, Versuch 2 für alle Tiere, Versuch 3 für alle Tiere, etc. Es sollte eine inter-Studie im Abstand von mindestens 2 Minuten. In regelmäßigen Abständen reinigen, am Pool, stellen Sie sicher, die Plattform ist vorhanden, und überprüfen Sie, dass die Wassertemperatur die gleiche ist. </li><li> Nachdem alle Tiere 12 Studien abgeschlossen haben, werden sie jedes Führen Sie eine Sonde Studie, in which die Plattform aus dem Pool entfernt. Die Sonde Studie wird durchgeführt, um das Tier das Verständnis der Plattform Lage zu überprüfen, und beobachten Sie die Strategie, dass das Tier folgt, wenn sie entdeckt die Plattform ist nicht da. Der Handler wird Freilassung des Tieres ab im Norden. Notieren Sie die Anzahl der das Tier kreuzt die Mitte des Pools während der 30 Sekunden. </li><li> Wenn alle die Sonde Tests abgeschlossen sind, abseits der Tiere trocken und Drain den Pool. </li></ol>

<ol>
	<li>de Bruin, J. P., Swinkels, W. A., de Brabander, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Response+learning+of+rats+in+a+Morris+water+maze:+involvement+of+the+medial+prefrontal+cortex.">Response learning of rats in a Morris water maze: involvement of the medial prefrontal cortex.</a> Behav. Brain Res. 85, 47-55 (1997).</li><li>Redish, A. D., Touretzky, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+the+hippocampus+in+solving+the+Morris+water+maze.">The role of the hippocampus in solving the Morris water maze.</a> Neural. Comput. 1, 73-111 (1998).</li><li>D'Hooge, R., De Deyn, P. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applications+of+the+Morris+water+maze+in+the+study+of+learning+and+memory.">Applications of the Morris water maze in the study of learning and memory.</a> Brain Res. Rev. 36, 60-90 (2001).</li><li>Kallai, J., Makany, T., Karadi, K., Jacobs, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+orientation+strategies+in+Morris-type+virtual+water+task+for+humans.">Spatial orientation strategies in Morris-type virtual water task for humans.</a> Behav. Brain Res. 159, 187-196 (2005).</li><li>Morris, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developments+of+a+water-maze+procedure+for+studying+spatial+learning+in+the+rat.">Developments of a water-maze procedure for studying spatial learning in the rat.</a> J. Neurosci. Methods. 11, 47-60 (1984).</li><li>Brandeis, R., Brandys, Y., Yehuda, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+the+Morris+Water+Maze+in+the+study+of+learning+and+memory.">The use of the Morris Water Maze in the study of learning and memory.</a> Int. J. Neurosci. 48, 26-69 (1989).</li><li>Kesner, R. P., Exans, R. B., Hunt, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Further+evidence+in+support+of+the+neurobiological+bases+of+an+attribute+model+of+memory:+role+of+the+hippocampus.">Further evidence in support of the neurobiological bases of an attribute model of memory: role of the hippocampus.</a> Int. J. Neurosci. 21-22, 21-22 (1987-8).</li><li>Jarrard, L. E., Okaichi, H., Steward, O., Goldschmidt, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+role+of+hippocampal+connections+in+the+performance+of+place+and+cue+tasks:+comparisons+with+damage+to+the+hippocampus.">On the role of hippocampal connections in the performance of place and cue tasks: comparisons with damage to the hippocampus.</a> Behav. Neurosci. 98, 946-954 (1984).</li><li>Hodges, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maze+procedures:+the+radial-arm+and+water+maze+compared.">Maze procedures: the radial-arm and water maze compared.</a> Cogn. Brain Res. 3, 167-181 (1996).</li><li>Shumake, J., Gonzalez-Lima, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain+systems+underlying+susceptibility+to+helplessness+and+depression.">Brain systems underlying susceptibility to helplessness and depression.</a> Behav. Cogn. Neurosci. Rev. 2, 198-221 (2003).</li><li>Owen, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiology+of+the+menstrual+cycle.">Physiology of the menstrual cycle.</a> Am. J. Clin. Nutr. 28, 333-338 (1975).</li><li>Everett, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurobiology+of+reproduction+in+the+female+rat.">Neurobiology of reproduction in the female rat.</a> Monogr. Endocrinol. 32, 1-133 (1989).</li><li>Warren, S. G., Humphreys, A. G., Juraska, J. M., Greenough, W. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LTP+varies+across+the+estrous+cycle:+enhanced+synaptic+plasticity+in+proestrus+rats.">LTP varies across the estrous cycle: enhanced synaptic plasticity in proestrus rats.</a> Brain Res. 703, 26-30 (1995).</li><li>Warren, S. G., Juraska, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+and+nonspatial+learning+across+the+rat+estrous+cycle.">Spatial and nonspatial learning across the rat estrous cycle.</a> Behav. Neurosci. 111, 259-266 (1997).</li></ol>

The authors have nothing to disclose.

Das Wasser Labyrinth Aufgabe war die Entwicklung von Morris 5. Diese Aufgabe kann in vielfältiger Weise zu untersuchen, Arbeitsspeicher, Referenz-Speicher und Aufgabe Strategie 6 verändert werden. Das hier beschriebene Verfahren besteht aus zwei wichtigen Variablen, die eine Abweichung von den anderen Versionen des Wassers Labyrinth darstellen: Vortraining und Tests an einem einzigen Tag. Vortraining - der Hippocampus ist in räumlicher / relationale Gedächtnis beteiligt 7-8. Das ...

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<td>SMART System</td>
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<td>This system is not a requirement &#x2013; there are many other behavioral systems on the market.</td>
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morris water maze experiment

Das Morris Water Maze ist weit verbreitet, um das räumliche Gedächtnis und das Lernen zu studieren. Die Tiere werden in einem Pool von Wasser, das gefärbte, opake mit pulverförmigen fettfreie Milch oder ungiftig Temperafarbe, wo sie zu einem versteckten entkommen Plattform schwimmen muss, ist gelegt. Weil sie in undurchsichtige Wasser sind, können die Tiere nicht sehen, die Plattform und kann nicht auf Duft verlassen, um den Fluchtweg zu finden. Stattdessen müssen sie auf externe / extra-maze Hinweise angewiesen. Da die Tiere mehr mit dieser Aufgabe vertraut sind, werden sie in der Lage, die Plattform noch schneller zu finden. Entwickelt von Richard G. Morris im Jahr 1984 hat dieses Paradigma zu einem der &quot;Goldstandard&quot; der Behavioral Neuroscience.

Watch this Scientific Journal Video about Morris Water Maze Experiment at JoVE.com

Morris Water Maze Experiment

Das Morris Water Maze ist eine allgemein anerkannte Werkzeug benutzt, um die Beteiligung des Hippocampus in einer Verhaltenstherapie Aufgabe zu dokumentieren.

Morris Water Maze-Experiment

The Morris water maze is widely used to study spatial memory and learning. Animals are placed in a pool of water that is colored opaque with powdered ...

Research

JoVE Journal

Biology

56.4K Aufrufe.  Michigan State University (MSU). Das Morris Water Maze ist eine allgemein anerkannte Werkzeug benutzt, um die Beteiligung des Hippocampus in einer Verhaltenstherapie Aufgabe zu dokumentieren.

Serie: Morris Water Maze Experiment

Testing Nicotine Tolerance in Aphids Using an Artificial Diet Experiment

Plants may upregulate the production of many different seconday metabolites in response to insect feeding. One of these metabolites, nicotine, is well know to have insecticidal properties. One response of tobacco plants to herbivory, or being gnawed upon by insects, is to increase the production of this neurotoxic alkaloid. Here, we will demonstrate how to set up an experiment to address this question of whether a tobacco-adapted strain of the green peach aphid, Myzus persicae, can tolerate higher levels of nicotine than the a strain of this insect that does not infest tobacco in the field.

In this video and assay is demonstrated that tests the tolerance of nicotine to two types of aphids one that infests tobacco plants in the field and one that does not.

Testen Nikotin Toleranz in Blattläuse mit einer künstlichen Diät Experiment

Plants may upregulate the production of many different seconday metabolites in response to insect feeding. One of these metabolites, nicotine, is well ...

Forschung

Biologie

Elevated Plus Maze for Mice

Although the mouse genome is now completely sequenced, the functions of most of the genes expressed in the brain are not known. The influence of a given gene on a specific behavior can be determined by behavioral analysis of mutant mice. If a target gene is expressed in the brain, behavioral phenotype of the mutant mice could elucidate the genetic mechanism of normal behaviors. The elevated plus maze test is one of the most widely used tests for measuring anxiety-like behavior. The test is based on the natural aversion of mice for open and elevated areas, as well as on their natural spontaneous exploratory behavior in novel environments. The apparatus consists of open arms and closed arms, crossed in the middle perpendicularly to each other, and a center area. Mice are given access to all of the arms and are allowed to move freely between them. The number of entries into the open arms and the time spent in the open arms are used as indices of open space-induced anxiety in mice. Unfortunately, the procedural differences that exist between laboratories make it difficult to duplicate and compare results among laboratories. Here, we present a detailed movie demonstrating our protocol for the elevated plus maze test. In our laboratory, we have assessed more than 90 strains of mutant mice using the protocol shown in the movie. These data will be disclosed as a part of a public database that we are now constructing.
Visualization of the protocol will promote better understanding of the details of the entire experimental procedure, allowing for standardization of the protocols used in different laboratories and comparisons of the behavioral phenotypes of various strains of mutant mice assessed using this test. 


The elevated plus maze test is one of the most widely used tests for measuring anxiety-like behavior in mice. Here, we present a movie showing the detailed procedures for conducting the test.

Elevated Plus Maze für Mäuse

Although the mouse genome is now completely sequenced, the functions of most of the genes expressed in the brain are not known. The influence of a given ...

Bioenergetic Profile Experiment using C2C12 Myoblast Cells

The ability to measure cellular metabolism and understand mitochondrial dysfunction, has enabled scientists worldwide to advance their research in understanding the role of mitochondrial function in obesity, diabetes, aging, cancer, cardiovascular function and safety toxicity.
Cellular metabolism is the process of substrate uptake, such as oxygen, glucose, fatty acids, and glutamine, and subsequent energy conversion through a series of enzymatically controlled oxidation and reduction reactions. These intracellular biochemical reactions result in the production of ATP, the release of heat and chemical byproducts, such as lactate and CO2 into the extracellular environment.
Valuable insight into the physiological state of cells, and the alteration of the state of those cells, can be gained through measuring the rate of oxygen consumed by the cells, an indicator of mitochondrial respiration - the Oxygen Consumption Rate - or OCR. Cells also generate ATP through glycolysis, i.e.: the conversion of glucose to lactate, independent of oxygen. In cultured wells, lactate is the primary source of protons. Measuring the lactic acid produced indirectly via protons released into the extracellular medium surrounding the cells, which causes acidification of the medium provides the Extra-Cellular Acidification Rate - or ECAR.
In this experiment, C2C12 myoblast cells are seeded at a given density in Seahorse cell culture plates. The basal oxygen consumption (OCR) and extracellular acidification (ECAR) rates are measured to establish baseline rates. The cells are then metabolically perturbed by three additions of different compounds (in succession) that shift the bioenergetic profile of the cell.
This assay is derived from a classic experiment to assess mitochondria and serves as a framework with which to build more complex experiments aimed at understanding both physiologic and pathophysiologic function of mitochondria and to predict the ability of cells to respond to stress and/or insults.

A description of a method for profiling mitochondrial function in cells is provided. The mitochondrial profile generated provides four parameters of mitochondrial function that can be measured in one experiment: basal respiration rate, ATP-linked respiration, proton leak, and reserve capacity.

Bioenergetische Profil Experiment mit C2C12 Myoblasten Cells

The ability to measure cellular metabolism and understand mitochondrial dysfunction, has enabled scientists worldwide to advance their research in ...

GC-based Detection of Aldononitrile Acetate Derivatized Glucosamine and Muramic Acid for Microbial Residue Determination in Soil

Quantitative approaches to characterizing microorganisms are crucial for a broader understanding of the microbial status and function within ecosystems. Current strategies for microbial analysis include both traditional laboratory culture-dependent techniques and those based on direct extraction and determination of certain biomarkers1, 2. Few among the diversity of microbial species inhabiting soil can be cultured, so culture-dependent methods introduce significant biases, a limitation absent in biomarker analysis.
The glucosamine, mannosamine, galactosamine and muramic acid have been well served as measures of both the living and dead microbial mass, of these the glucosamine (most abundant) and muramic acid (uniquely from bacterial cell) are most important constituents in the soil systems3, 4. However, the lack of knowledge on the analysis restricts the wide popularization among scientific peers. Among all existing analytical methods, derivatization to aldononitrile acetates followed by GC-based analysis has emerged as a good option with respect to optimally balancing precision, sensitivity, simplicity, good chromatographic separation, and stability upon sample storage5.
Here, we present a detailed protocol for a reliable and relatively simple analysis of glucosamine and muramic acid from soil after their conversion to aldononitrile acetates. The protocol mainly comprises four steps: acid digestion, sample purification, derivatization and GC determination. The step-by-step procedure is modified according to former publications6, 7. In addition, we present a strategy to structurally validate the molecular ion of the derivative and its ion fragments formed upon electron ionization. We applied GC-EI-MS-SIM, LC-ESI-TOF-MS and isotopically labeled reagents to determine the molecular weight of aldononitrile acetate derivatized glucosamine and muramic acid; we used the mass shift of isotope-labeled derivatives in the ion spectrum to investigate ion fragments of each derivatives8. In addition to the theoretical elucidation, the validation of molecular ion of the derivative and its ion fragments will be useful to researchers using &delta;13C or ion fragments of these biomarkers in biogeochemical studies9, 10.

We describe a method protocol for the GC-based analysis of the aldonitrile acetate derivatives of glucosamine and muramic acid extracted from soil. For elucidation of the chemical mechanism, we also present a strategy to confirm the structure of the derivative and the ion fragments formed upon electron ionization.

GC-basierte Erkennung von Aldononitrile Acetat Derivatisierte Glucosamin und Muraminsäure für Mikrobielle Rückstandsbestimmung im Boden

Quantitative approaches to characterizing microorganisms are crucial for a broader understanding of the microbial status and function within ecosystems. ...

Protease- and Acid-catalyzed Labeling Workflows Employing 18O-enriched Water

Stable isotopes are essential tools in biological mass spectrometry. Historically, 18O-stable isotopes have been extensively used to study the catalytic mechanisms of proteolytic enzymes1-3. With the advent of mass spectrometry-based proteomics, the enzymatically-catalyzed incorporation of 18O-atoms from stable isotopically enriched water has become a popular method to quantitatively compare protein expression levels (reviewed by Fenselau and Yao4, Miyagi and Rao5 and Ye et al.6). 18O-labeling constitutes a simple and low-cost alternative to chemical (e.g. iTRAQ, ICAT) and metabolic (e.g. SILAC) labeling techniques7. Depending on the protease utilized, 18O-labeling can result in the incorporation of up to two 18O-atoms in the C-terminal carboxyl group of the cleavage product3. The labeling reaction can be subdivided into two independent processes, the peptide bond cleavage and the carboxyl oxygen exchange reaction8. In our PALeO (protease-assisted labeling employing 18O-enriched water) adaptation of enzymatic 18O-labeling, we utilized 50% 18O-enriched water to yield distinctive isotope signatures. In combination with high-resolution matrix-assisted laser desorption ionization time-of-flight tandem mass spectrometry (MALDI-TOF/TOF MS/MS), the characteristic isotope envelopes can be used to identify cleavage products with a high level of specificity. We previously have used the PALeO-methodology to detect and characterize endogenous proteases9 and monitor proteolytic reactions10-11. Since PALeO encodes the very essence of the proteolytic cleavage reaction, the experimental setup is simple and biochemical enrichment steps of cleavage products can be circumvented. The PALeO-method can easily be extended to (i) time course experiments that monitor the dynamics of proteolytic cleavage reactions and (ii) the analysis of proteolysis in complex biological samples that represent physiological conditions. PALeO-TimeCourse experiments help identifying rate-limiting processing steps and reaction intermediates in complex proteolytic pathway reactions. Furthermore, the PALeO-reaction allows us to identify proteolytic enzymes such as the serine protease trypsin that is capable to rebind its cleavage products and catalyze the incorporation of a second 18O-atom. Such "double-labeling" enzymes can be used for postdigestion 18O-labeling, in which peptides are exclusively labeled by the carboxyl oxygen exchange reaction. Our third strategy extends labeling employing 18O-enriched water beyond enzymes and uses acidic pH conditions to introduce 18O-stable isotope signatures into peptides.

Protease- and Acid-catalyzed Labeling Workflows Employing 18O-enriched Water

Stable isotope labeling workflows employing 18O-enriched water (LeO-workflows) are versatile tools for quantitative and qualitative proteomics studies. In protease-assisted (PALeO) workflows, 18O-atoms are introduced by proteolytic cleavage and carboxyl oxygen exchange reactions mediated by proteases. In the acid-catalyzed (ALeO) workflow, 18O-atoms are introduced by carboxyl oxygen exchange at low pH.

Protease-und Acid-katalysierten Labeling Workflows beschäftigt<sup&gt; 18</sup&gt; O-angereichertem Wasser

Stable isotopes are essential tools in biological mass spectrometry. Historically, 18O-stable isotopes have been extensively used to study the catalytic ...

A Semi-quantitative Approach to Assess Biofilm Formation Using Wrinkled Colony Development

Biofilms, or surface-attached communities of cells encapsulated in an extracellular matrix, represent a common lifestyle for many bacteria. Within a biofilm, bacterial cells often exhibit altered physiology, including enhanced resistance to antibiotics and other environmental stresses 1. Additionally, biofilms can play important roles in host-microbe interactions. Biofilms develop when bacteria transition from individual, planktonic cells to form complex, multi-cellular communities 2. In the laboratory, biofilms are studied by assessing the development of specific biofilm phenotypes. A common biofilm phenotype involves the formation of wrinkled or rugose bacterial colonies on solid agar media 3. Wrinkled colony formation provides a particularly simple and useful means to identify and characterize bacterial strains exhibiting altered biofilm phenotypes, and to investigate environmental conditions that impact biofilm formation. Wrinkled colony formation serves as an indicator of biofilm formation in a variety of bacteria, including both Gram-positive bacteria, such as Bacillus subtilis 4, and Gram-negative bacteria, such as Vibrio cholerae 5, Vibrio parahaemolyticus 6, Pseudomonas aeruginosa 7, and Vibrio fischeri 8.
The marine bacterium V. fischeri has become a model for biofilm formation due to the critical role of biofilms during host colonization: biofilms produced by V. fischeri promote its colonization of the Hawaiian bobtail squid Euprymna scolopes 8-10. Importantly, biofilm phenotypes observed in vitro correlate with the ability of V. fischeri cells to effectively colonize host animals: strains impaired for biofilm formation in vitro possess a colonization defect 9,11, while strains exhibiting increased biofilm phenotypes are enhanced for colonization 8,12. V. fischeri therefore provides a simple model system to assess the mechanisms by which bacteria regulate biofilm formation and how biofilms impact host colonization.
In this report, we describe a semi-quantitative method to assess biofilm formation using V. fischeri as a model system. This method involves the careful spotting of bacterial cultures at defined concentrations and volumes onto solid agar media; a spotted culture is synonymous to a single bacterial colony. This 'spotted culture' technique can be utilized to compare gross biofilm phenotypes at single, specified time-points (end-point assays), or to identify and characterize subtle biofilm phenotypes through time-course assays of biofilm development and measurements of the colony diameter, which is influenced by biofilm formation. Thus, this technique provides a semi-quantitative analysis of biofilm formation, permitting evaluation of the timing and patterning of wrinkled colony development and the relative size of the developing structure, characteristics that extend beyond the simple overall morphology.

We provide a simple, semi-quantitative method to investigate biofilm formation in vitro. This method takes advantage of the Zeiss stemi 2000-C Dissecting Microscope (with camera attachment) to monitor both the timing and pattern of biofilm formation, as assessed by the development of wrinkled colonies.

Ein semi-quantitativer Ansatz, um die Biofilmbildung Mit Wrinkled Colony Entwicklung zu bewerten

Biofilms, or surface-attached communities of cells encapsulated in an extracellular matrix, represent a common lifestyle for many bacteria. Within a ...

Isolation and Culture of Neonatal Mouse Cardiomyocytes

Cultured neonatal cardiomyocytes have long been used to study myofibrillogenesis and myofibrillar functions. Cultured cardiomyocytes allow for easy investigation and manipulation of biochemical pathways, and their effect on the biomechanical properties of spontaneously beating cardiomyocytes.	
The following 2-day protocol describes the isolation and culture of neonatal mouse cardiomyocytes. We show how to easily dissect hearts from neonates, dissociate the cardiac tissue and enrich cardiomyocytes from the cardiac cell-population. We discuss the usage of different enzyme mixes for cell-dissociation, and their effects on cell-viability. The isolated cardiomyocytes can be subsequently used for a variety of morphological, electrophysiological, biochemical, cell-biological or biomechanical assays. We optimized the protocol for robustness and reproducibility, by using only commercially available solutions and enzyme mixes that show little lot-to-lot variability. We also address common problems associated with the isolation and culture of cardiomyocytes, and offer a variety of options for the optimization of isolation and culture conditions.

Primary mouse cardiomyocyte cultures are one of the pivotal tools for the investigation of myofibrillar organization and function. The following protocol describes the isolation and culture of primary cardiomyocytes from neonatal mouse hearts. The resulting cardiomyocyte cultures may be subsequently used for a variety of biomechanical, biochemical and cell-biological assays.

Isolation und Kultur der Neugeborenen-Maus-Kardiomyozyten

Cultured neonatal cardiomyocytes have long been used to study  myofibrillogenesis and myofibrillar functions. Cultured cardiomyocytes allow  for easy ...

High Content Screening Analysis to Evaluate the Toxicological Effects of Harmful and Potentially Harmful Constituents (HPHC)

Cigarette smoke (CS) is a major risk factor for cardiovascular and lung diseases. Because CS is a complex aerosol containing more than 7,000 chemicals1 it is challenging to assess the contributions of individual constituents to its overall toxicity. Toxicological profiles of individual constituents as well as mixtures can be however established in vitro, by applying high through-put screening tools, which enable the profiling of Harmful and Potentially Harmful Constituents (HPHCs) of tobacco smoke, as defined by the U.S. Food and Drug Administration (FDA).2
For an initial assessment, an impedance-based instrument was used for a real-time, label-free assessment of the compound&#39;s toxicity. The instrument readout relies on cell adhesion, viability and morphology that all together provide an overview of the cell status. A dimensionless parameter, named cell index, is used for quantification. A set of different staining protocols was developed for a fluorescence imaging-based investigation and a HCS platform was used to gain more in-depth information on the kind of cytotoxicity elicited by each HPHC.
Of the 15 constituents tested, only five were selected for HCS-based analysis as they registered a computable LD50 (&#60; 20 mM). These included 1-aminonaphtalene, Arsenic (V), Chromium (VI), Crotonaldehyde and Phenol. Based on their effect in the HCS, 1-aminonaphtalene and Phenol could be identified to induce mitochondrial dysfunction, and, together with Chromium (VI) as genotoxic based on the increased histone H2AX phosphorylation. Crotonaldehyde was identified as an oxidative stress inducer and Arsenic as a stress kinase pathway activator.
This study demonstrates that a combination of impedance-based and HCS technologies provides a robust tool for in vitro assessment of CS constituents.

High Content Screening Analysis to Evaluate the Toxicological Effects of Harmful and Potentially Harmful Constituents HPHC

The objective of the study was to assess the biological impact of 15 cigarette smoke constituents using a combination of an impedance-based real time cell analyzer and a high-content screening (HCS)-based platform for toxicological assessment in vitro. This study provides information on effective doses, toxicity and modes of action of the tested compounds.

High Content Screening-Analyse die toxikologischen Wirkungen von gefährlichen und potenziell gefährlichen Inhaltsstoffe (HPHC) zur Bewertung

Cigarette smoke (CS) is a major risk factor for cardiovascular and lung diseases. Because CS is a complex aerosol containing more than 7,000 chemicals1 it ...

Isolation of Mouse Epidermal Keratinocytes and Their In Vitro Clonogenic Culture

The protocol described here is a reliable method of harvesting primary keratinocytes from adult female mice (54 &#177; 2 days old) yielding approximately 30 x 106 viable cells per mouse. Primary adult mouse keratinocytes are harvested from the dorsal skin of female mice. Male mice (~6 weeks old) can be used for keratinocyte harvesting depending on the requirements of the experiment. Euthanized mice are shaved and sterilized with serial washes in povidone iodine and ethanol solutions (70% alcohol). After disinfecting the mice, the dorsal skin is removed and the subcutaneous fat and muscle are removed with a scalpel and discarded. The skins are cut into small pieces and treated with a mild, low temperature trypsinization to detach the lower dermis from the epidermis. The scraped epidermises are stirred at low speed, filtered to remove the hairs, counted, and re-suspended in culture medium. This method provides an excellent single cell suspension of highly culturable cells for many downstream applications.

The goal of this protocol is to isolate epidermal keratinocytes from the dorsal skin of adult mice for a variety of downstream applications such as molecular biology, biochemistry, fluorescence activated cell sorting, and primary in vitro uses (e.g., clonogenic keratinocytes).

Isolierung von Maus epidermalen Keratinozyten und ihrer In-Vitro-Clonogenen Kultur

The protocol described here is a reliable method of harvesting primary keratinocytes from adult female mice (54 &#177; 2 days old) yielding approximately ...

Evidence for EpCAM and Cytokeratin Expressing Epithelial Cells in Normal Human and Murine Blood and Bone Marrow

Epithelial cells have been identified in the blood and bone marrow of patients with cancer and other diseases. However, the presence of normal epithelial cells in the blood and bone marrow of healthy individuals has yet to be identified in a consistent way. Presented here is a reproducible method for isolating epithelial cells from healthy human and murine blood and bone marrow (BM) using flow cytometry and immunofluorescence (IF) microscopy. Epithelial cells in healthy individuals were first identified and isolated via flow cytometry using epithelial cell adhesion molecule (EpCAM). These EpCAM+ cells were confirmed to express keratin using immunofluorescence microscopy in Krt1-14;mTmG transgenic mice. Human blood samples had 0.18% &#177; 0.0004 EpCAM+ cells (SEM; n=7 biological replicates, 4 experimental replicates). In human BM, 3.53% &#177; 0.006 (SEM; n=3 biological replicates, 4 experimental replicates) of mononuclear cells were EpCAM+. In mouse blood, EpCAM+ cells constituted 0.45% &#177; 0.0006 (SEM; n=2 biological replicates, 4 experimental replicates), and in mouse BM, 5.17% &#177; 0.001 (SEM; n=3 biological replicates, 4 experimental replicates) were EpCAM+. In mice, all the EpCAM+ cells were immunoreactive to pan-cytokeratin, as determined by IF microscopy. Results were confirmed using Krt1-14;mTmG transgenic mice, with low (8.6 native GFP+ cells per 106 cells analyzed; 0.085% of viable cells), but significant numbers (p &#60; 0.0005) of GFP+ cells present in normal murine BM, that were not the result of randomness compared with multiple negative controls. Further, EpCAM+ cells in mouse blood were more heterogeneous than CD45+ cells (0.58% in BM; 0.13% in blood). These observations conclude that cells expressing cytokeratin proteins are reproducibly detectable among mononuclear cells from human and murine blood and BM. We demonstrate a method of tissue harvesting, flow cytometry, and immunostaining that can be used to identify and determine the function of these pan-cytokeratin epithelial cells in healthy individuals.

This paper presents a reproducible method with new findings on the presence of epithelial cells in normal human and mouse blood and bone marrow using flow cytometry and immunofluorescence microscopy. Krt1-14;mTmG transgenic mice were used as an in vivo method to confirm these findings.

Evidenz für EpCAM und Zytokeratin-exprimierende Epithelzellen in normalem menschlichem und murinem Blut und Knochenmark

Epithelial cells have been identified in the blood and bone marrow of patients with cancer and other diseases. However, the presence of normal epithelial ...